Supplementary Figure 2: Epithelial organisation on FAM40A, FAM40B and STRN3 depletion in human cancer cells.

(a,b) siRNA depleted A431 cells plated on collagen-I/matrigels. Cells were fixed 72 h post-transfection and stained (a) against pS19-MLC (green) and (b) pT18S19-MLC (blue) and E-Cadherin (green). F-actin is stained with Phalloidin-TRITC (red). (c) siRNA knockdown efficiency +/s.e.m of FAM40A and FAM40B mRNA in A431 cells. Immunoblotting analysis of FAM40B protein levels on FAM40B depletion. (d) Deconvolution of siRNA oligos in A431 cells plated on collagen-I/matrigel. Cells were stained 72 h post-transfection against pS19-MLC2 (white) and F-actin (red). Quantification of A431 cell area +/−s.e.m (upper histogram, n = fields of cells; Ctr, 19; siFAM40A_2, 9; siFAM40A_5, 6; siFAM40A_7, 5; siFAM40B-3, 12; siFAM40B-6, 6; siFAM40B-7, 7) and mean fluorescence pS19-MLC2 intensities +/−s.e.m (lower histogram, n = fields of cells; Ctr, 10; siFAM40A_2, 6; siFAM40A_5, 4; siFAM40A_7, 4; siFAM40B-3, 6; siFAM40B-6, 6; siFAM40B-7,6). (e) Quantification of mean fluorescence cortical intensities of pS19-MLC2 (left panel, n = cells; Ctr, 33; siFAM40A, 46; siFAM40B, 47) and pERM (right panel, n = cells; Ctr, 29; siFAM40A, 35; siFAM40B, 43) in MDA-MB-231 cells. Box and whiskers graph: Line = Median, Box = distribution of 50% of values, whiskers = min. to max. (f) siRNA depleted MDA-MB-231 cells were fixed 72 h post-transfection. Focal adhesions were stained with phospho-tyrosine antibody (cl. 4G10), and thin sections of the focal adhesions in the basal plasma membrane were acquired using confocal microscopy. Morphometric analysis was performed on individual cells. Two independent experiments were conducted and 20–25 cells quantified. n = focal adhesions; Ctr, 468; siFAM40A, 401; siFAM40B, 558). (g,h) Cell surface expression of integrin β1 (ITGB1) using flow cytometry was performed on MDA-MB-231 cells 72 h post-transfection. Cell surface expression of ITGB1 +/−s.d. was detected using the HUTS-21 antibody detecting the active form with or without the presence of 0.5 mM MnCl₂ to activate all integrins on the cell surface (n = 3 experiments). (i) Quantification of pS19-MLC2 and cytosolic GFP co-localisation + /s.e.m. (n = cells; siCtr, 25; siFAM40A, 16; siFAM40B, 17). (j) siRNA depleted MDA-MB-231-Ezrin-GFP cells were fixed 72 h post-transfection. Cells were stained for MyoIIA (red), and thin 150 nm sections were acquired using Structured Illumination Microscopy (left panels). The spatial correlation (r) between Ezrin-GFP (green) and MyoIIA (red) was then determined on FAM40A and FAM40B depletion and compared to control cells (right panels). Statistical test were performed using 1way ANOVA, Sidak’s multiple comparison test, ^∗P < 0.05,^∗∗P < 0.01,^∗∗∗P < 0.001. All experiments were conducted at least 3 independent times. Scale bars, 10 μm, unless indicated otherwise.